Biophysical Chemistry of Proteins

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Engelbert Buxbaum

Biophysical Chemistry
of Proteins
An Introduction to Laboratory Methods

ABC
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Engelbert Buxbaum
Ross University School of Medicine
P.O. Box 266
Biochemistry
Portsmouth Campus
266 Roseau
Dominica
engelbert

ISBN 978-1-4419-7250-7
e-ISBN 978-1-4419-7251-4
DOI 10.1007/978-1-4419-7251-4

Springer New York Dordrecht Heidelberg London
© Springer Science+Business Media, LLC 2011
All rights reserved. This work may not be translated or copied in whole or in part without the written
permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York,
NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in
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or by similar or dissimilar methodology now known or hereafter developed is forbidden.
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Preface

During undergraduate courses in biochemistry you learned what proteins do as
enzymes, receptors, hormones, motors or structural components. The more interesting question, how proteins can achieve all these functions, is usually asked only
in graduate courses, and in many cases it is a topic of ongoing research.
Here I want to present an overview of the methods used in such research projects,
their possible applications, and their limitations. I have limited the presentation to
a level where a general background in chemistry, physics, and mathematics is sufficient to follow the discussion. Quantum mechanics, where required, is treated in a
purely qualitative manner. A good understanding of protein structure and enzymology is required, but these topics I have covered in a separate volume [44].
Apart from graduate training in protein science this book should also be useful
as a reference for people who work with proteins.
After studying this book you should be able to collaborate with workers who have
the required instruments and use these methods routinely. You should also be able
to understand papers which make use of such methods. However, before embarking

on independent research using these methods you are directed to the literature cited
for a more in-depth, more quantitative coverage.
This book focuses on the biophysical chemistry of proteins. The use of nucleic
acid-based methods [360], although in many cases very relevant and informative, is
outside the scope of this text. Also only hinted at are modern approaches to computational biochemistry [20, 180, 231]. In the end, the models derived from such
techniques have to be verified by experiments. If this book stimulates such studies,
it has served its purpose.

Acknowledgements
I wish to thank all my students, friends, and colleagues who have given me their
support and suggestions for this text, and who have gone through the arduous
task of proof-reading. All remaining errors are, of course, mine. Please report
any errors found and any suggestions for improvement to me (mailto://engelbert
).
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Preface

A big “thank you” goes to all those who have made software freely available, or
who maintain repositories of information on the internet. Without your dedication,
this book would not have been possible.
Portsmouth, Dominica

Engelbert Buxbaum


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Contents

Part I Analytical Techniques
1

Microscopy .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
1.1 Optical Foundations of Microscopy .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
1.1.1
K OHLER-Illumination .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
1.1.2 The Role of Diffraction . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
1.1.3 The Importance of the Numerical Aperture Na . . . . . . . . . . . . .
1.1.4 Homogeneous Immersion . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
1.1.5 Lens Aberrations.. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
1.1.6 Special Methods in Light Microscopy .. . . . .. . . . . . . . . . . . . . . . .
1.2 The Electron Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
1.2.1 Transmission Electron Microscopy .. . . . . . . .. . . . . . . . . . . . . . . . .
1.2.2 Scanning Electron Microscopy . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
1.2.3 Freeze Fracture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
1.3 Other Types of Microscopes .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
1.3.1 The Atomic Force Microscope .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
1.3.2 The Scanning Tunnelling Microscope .. . . . .. . . . . . . . . . . . . . . . .
1.3.3 The Scanning Near-Field Optical Microscope . . . . . . . . . . . . . .

3
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2

Single Molecule Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 23
2.1 Laser Tweezers and Optical Trapping .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 23

3

Preparation of Cells and Tissues for Microscopy . . . . . . . .. . . . . . . . . . . . . . . . .
3.1 Fixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
3.2 Embedding and Cutting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
3.3 Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
3.4 Laser Precision Catapulting.. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .

4

Principles of Optical Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 27
4.1 Resonant Interaction of Molecules and Light .. . . . . . .. . . . . . . . . . . . . . . . . 27

4.2 The Evanescent Wave . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 29

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5

Photometry .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
5.1 Instrumentation .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
5.2
L AMBERT–B EER’s Law . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
5.2.1 The Isosbestic Point . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
5.3 Environmental Effects on a Spectrum .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .

33
33
33
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36

6

Fluorimetry.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.1 Fluorescent Proteins .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.2 Lanthanoid Chelates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.2.1 Quantum Dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.3 Fluorescence Quenching .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.3.1 Dynamic Quenching .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.3.2 Static Quenching.. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
ă
Resonance Energy Transfer . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.4
F ORSTER
6.4.1 Handling Channel Spillover .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.4.2 Homogeneous FRET Assays . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.4.3 Problems to Be Aware Of . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.4.4 Fluorescence Complementation.. . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.4.5 Pulsed Excitation with Multiple Wavelengths.. . . . . . . . . . . . . .
6.5 Photoinduced Electron Transfer . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.6 Fluorescence Polarisation .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.6.1 Static Fluorescence Polarisation . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.6.2 Application.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.7 Time-Resolved Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.7.1 Fluorescence Autocorrelation .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
6.7.2 Dynamic Fluorescence Polarisation . . . . . . . .. . . . . . . . . . . . . . . . .
6.8 Photo-bleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .

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7

Chemiluminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
7.1 Chemiluminescent Compounds . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
7.2 Assay Conditions .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
7.3 Electrochemiluminescence.. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .

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59
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8

Electrophoresis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
8.1 Movement of Poly-ions in an Electrical Field . . . . . . .. . . . . . . . . . . . . . . . .
8.1.1 Influence of Running Conditions . . . . . . . . . . .. . . . . . . . . . . . . . . . .
8.2 Electrophoretic Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
8.2.1 Techniques of Historic Interest . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
8.2.2 Gel Electrophoresis .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
8.2.3 Free-Flow Electrophoresis . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
8.2.4 Native Electrophoresis . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
8.2.5 Denaturing Electrophoresis . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
8.2.6 Blue Native PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
8.2.7 CTAB-Electrophoresis . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
8.2.8 Practical Hints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .

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8.2.9
8.2.10
8.2.11
8.2.12
9

IEF and 2D-electrophoresis . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
Elution of Proteins from Electrophoretic Gels . . . . . . . . . . . . . .
Gel Staining Procedures .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
Capillary Electrophoresis . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .

81
90
90
94

Immunological Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 97
9.1 Production of Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 97
9.1.1 Isolation from Animals . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 97
9.1.2 Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .100
9.1.3 Artificial Antibodies .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .101
9.1.4 Aptamers .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .102

9.2 Immunodiffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .103
9.3 Immunoelectrophoretic Methods . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .104
9.4 RIA, ELISA and Immuno-PCR . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .104
9.4.1 RIA .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .105
9.4.2 ELISA .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .105
9.4.3 Immuno-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .107
9.5 Methods that Do Not Require Separation of Bound
and Unbound Antigen .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .107
9.5.1 Microwave and Surface Plasmon Enhanced Techniques . . .110
9.6 Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .110
9.6.1 Western Blots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .111
9.6.2 Dot Blots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .114
9.6.3 Total Protein Staining of Blots . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .114
9.6.4 Immunostaining of Blots . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .115
9.7 Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .117
9.8 Immunomicroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .117
9.9 Fluorescent Cell Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .119
9.10 Protein Array Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .120

10 Isotope Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .123
10.1 Radioisotopes .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .123
10.1.1 The Nature of Radioactivity.. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .124
10.1.2 Measuring “-Radiation . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .126
10.1.3 Measuring ”-Radiation .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .131
10.2 Stable Isotopes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .131
Part II Purification of Proteins
11 Homogenisation and Fractionisation of Cells and Tissues . . . . . . . . . . . . . . .135
11.1 Protease Inhibitors.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .136
12 Isolation of Organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .141


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13 Precipitation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .143
13.1 Salts . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .143
13.2 Organic Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .145
13.3 Heat . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .146
14 Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .147
14.1 Chromatographic Methods.. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .147
14.2 Theory of Chromatography .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .152
14.2.1 The C RAIG-Distribution . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .152
14.2.2 Characterising Matrix–Solute Interaction . .. . . . . . . . . . . . . . . . .155
14.2.3 The Performance of Chromatographic Columns .. . . . . . . . . . .157
14.3 Strategic Considerations in Protein Purification . . . . .. . . . . . . . . . . . . . . . .161
14.3.1 Example: Purification of Nucleotide-free
Hsc70 From Mung Bean Seeds . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .161
15 Membrane Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .163
15.1 Structure of Lipid/Water Systems . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .163
15.2 Physicochemistry of Detergents . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .166
15.2.1 Detergent Partitioning into Biological Membranes . . . . . . . . .171
15.3 Detergents in Membrane Protein Isolation .. . . . . . . . . .. . . . . . . . . . . . . . . . .174
15.3.1 Functional Solubilisation of Proteins .. . . . . .. . . . . . . . . . . . . . . . .174
15.3.2 Isolation of Solubilised Proteins . . . . . . . . . . . .. . . . . . . . . . . . . . . . .176
15.3.3 Reconstitution of Proteins into Model Membranes . . . . . . . . .177
15.4 Developing a Solubilisation Protocol . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .179
15.5 Membrane Lipids: Preparation, Analysis and Handling .. . . . . . . . . . . . .181

15.5.1 Measurements with Lipids and Membranes .. . . . . . . . . . . . . . . .181
16 Determination of Protein Concentration.. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .183
17 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .187
17.1 Cell Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .188
17.1.1 Contamination of Cell Cultures . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .189
Part III Protein Modification and Inactivation
18 General Technical Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .193
18.1 Determining the Specificity of Labelling . . . . . . . . . . . .. . . . . . . . . . . . . . . . .194
18.2 Kinetics of Enzyme Modification . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .194
19 Amine-Reactive Reagents .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .199
20 Thiol and Disulphide Reactive Reagents .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .205
20.1 Cystine Reduction .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .207

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21 Reagents for Other Groups .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .209
21.1 The Alcoholic OH-Group .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .209
21.2 The Phenolic OH-Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .210
21.3 Carboxylic Acids .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .212
21.4 Histidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .213
21.4.1 Tryptophan .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .213
21.4.2 Arginine .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .215
21.4.3 Methionine .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .215
22 Cross-linkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .219
22.1 Reversible Cross-linkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .219

22.2 Trifunctional Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .220
23 Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .223
23.1 Radio-labelling of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .223
23.2 Photo-reactive Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .224
23.3 Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .224
23.4 Particle Based Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .226
23.4.1 Colloidal Gold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .226
23.4.2 Magnetic Separation .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .227
24 Spontaneous Reactions in Proteins . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .229
24.1 Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .229
24.1.1 Racemisation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .229
24.1.2 Oxidation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .230
24.1.3 Amyloid-Formation . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .230
24.2 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .232
Part IV

Protein Size and Shape

25 Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .237
25.1 Theory of Centrifugation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .238
25.1.1 Spherical Particles . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .238
25.1.2 Non-spherical Particles . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .240
25.1.3 Determination of Molecular Mass . . . . . . . . . .. . . . . . . . . . . . . . . . .241
25.1.4 Pelleting Efficiency of a Rotor .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . .244
25.2 Centrifugation Techniques .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .245
25.3 Rotor-Types .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .246
25.4 Types of Centrifuges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .247
25.5 Determination of the Partial Specific Volume.. . . . . . .. . . . . . . . . . . . . . . . .248
26 Osmotic Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .251
26.1 Dialysis of Charged Species: The D ONNAN-Potential . . . . . . . . . . . . . . .252

27 Diffusion . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .255

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28 Viscosity . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .257
29 Non-resonant Interactions with Electromagnetic Waves .. . . . . . . . . . . . . . . .261
29.1 Laser Light Scattering.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .261
29.1.1 Static Light Scattering .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .261
29.1.2 Dynamic Light Scattering .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .263
29.1.3 Quasi-elastic Scattering . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .264
29.1.4 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .265
29.2 Small Angle X-ray Scattering SAXS. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .265
29.3 Neutron Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .265
29.4 Radiation Inactivation .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .266
Part V

Protein Structure

30 Protein Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .271
30.1 Edman Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .271
30.1.1 Problems that May Be Encountered . . . . . . . .. . . . . . . . . . . . . . . . .272
30.1.2 Sequxencing in the Genomic Age . . . . . . . . . .. . . . . . . . . . . . . . . . .273
30.2 Mass Spectrometry .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .274
30.2.1 Ionisers .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .274
30.2.2 Analysers (See Fig. 30.6) .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .277

30.2.3 Determination of Protein Molecular Mass
by Mass Spectrometry .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .280
30.2.4 Tandem Mass Spectrometry .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .281
30.2.5 Protein Sequencing by Tandem MS . . . . . . . .. . . . . . . . . . . . . . . . .282
30.2.6 Digestion of Proteins . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .284
30.2.7 Ion–Ion Interactions . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .284
30.3 Special Uses of MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .286
30.3.1 Disease Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .287
30.3.2 Shotgun Sequencing of Proteins . . . . . . . . . . . .. . . . . . . . . . . . . . . . .287
30.4 Characterising Post-translational Modifications . . . . .. . . . . . . . . . . . . . . . .287
30.4.1 Ubiquitinated Proteins .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .287
30.4.2 Methylation, Acetylation and Oxidation . . .. . . . . . . . . . . . . . . . .287
30.4.3 Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .288
31 Synthesis of Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .289
32 Protein Secondary Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .291
32.1 Circular Dichroism Spectroscopy . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .291
32.2 Infrared Spectroscopy .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .294
32.2.1 Attenuated Total Internal Reflection IR-Spectroscopy.. . . . .296
32.2.2 Fourier-Transform IR-Spectroscopy .. . . . . . .. . . . . . . . . . . . . . . . .296
32.2.3 IR-Spectroscopy of Proteins . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .297
32.2.4 Measuring Electrical Fields in Enzymes:
The S TARK-effect . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .301
32.3 Raman-Spectroscopy .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .302

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33 Structure of Protein–Ligand Complexes .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .303
33.1 Electron-Spin Resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .303
33.1.1 Factors to Be Aware Of.. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .304
33.1.2 Natural ESR Probes with Single Electrons .. . . . . . . . . . . . . . . . .305
33.1.3 Stable Free Radical Spin Probes . . . . . . . . . . . .. . . . . . . . . . . . . . . . .305
33.1.4 Hyperfine Splitting: ENDOR-Spectroscopy.. . . . . . . . . . . . . . . .307
33.1.5 ESR of Triplet States . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .307
33.2 X-ray Absorbtion Spectroscopy . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .307
33.2.1 Production of X-rays . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .307
33.2.2 Absorbtion of X-rays . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .308
34 3-D Structures.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .309
34.1 Nuclear Magnetic Resonance .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .309
34.1.1 Theory of 1-D NMR . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .309
34.1.2 B OLTZMANN-Distribution of Spins . . . . . . . .. . . . . . . . . . . . . . . . .310
34.1.3 Parameters Detected by 1-D NMR . . . . . . . . .. . . . . . . . . . . . . . . . .312
34.1.4 NMR of Proteins, Multi-dimensional NMR. . . . . . . . . . . . . . . . .314
34.1.5 Solid State NMR. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .318
34.2 Computerised Structure Refinement . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .319
34.2.1 Energy Minimisation . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .319
34.2.2 Molecular Dynamics . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .320
34.2.3 Monte Carlo Simulations .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .320
34.2.4 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .321
34.3 X-ray Crystallography of Proteins.. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .321
34.3.1 Crystallisation of Proteins .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .322
34.3.2 Sparse Matrix Approaches to Experimental
Design: The TAGUCHI-method . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .330
34.3.3 X-Ray Structure Determination .. . . . . . . . . . . .. . . . . . . . . . . . . . . . .331
34.3.4 Other Diffraction Techniques . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .339
34.4 Electron Microscopy of 2-D Crystals . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .341

35 Folding and Unfolding of Proteins .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .343
35.1 Inserting Proteins into a Membrane . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .343
35.2 Change of Environment .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .344
35.2.1 Standard Conditions for Experiments . . . . . .. . . . . . . . . . . . . . . . .346
35.3 The Chevron-Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .346
35.3.1 Unfolding by Pulse Proteolysis and Western-Blot . . . . . . . . . .347
35.3.2 Non-linear Chevron-Plots . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .348
35.3.3 Unfolding During Electrophoresis .. . . . . . . . .. . . . . . . . . . . . . . . . .348
35.3.4 Membrane Proteins .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .349
35.4 The Double-Jump Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .349
35.5 Hydrogen Exchange .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .349
35.6 Differential Scanning Calorimetry . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .350
35.7 The Protein Engineering Method .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .350

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Part VI Enzyme Kinetics
36 Steady-State Kinetics .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .355
36.1 Assays of Enzyme Activity . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .356
36.1.1 The Coupled Spectrophotometric Assay of WARBURG . . . .357
36.2 Environmental Influences on Enzymes .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .359
36.2.1 pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .359
36.2.2 Ionic Strength .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .359
36.2.3 Temperature .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .360
36.3 Synergistic and Antagonistic Interactions .. . . . . . . . . . .. . . . . . . . . . . . . . . . .361

36.3.1 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .361
36.3.2 The Isobologram .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .361
36.3.3 Predicting the Effect for Combinations
of Independently Acting Agents . . . . . . . . . . . .. . . . . . . . . . . . . . . . .362
36.4 Stereoselectivity .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .364
37 Leaving the Steady State: Analysis of Progress Curves .. . . . . . . . . . . . . . . . .367
38 Reaction Velocities .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .369
38.1 Near Equilibrium Higher Order Reactions
can be Treated as First Order . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .369
38.2 Continuous Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .370
38.3 Quenched Flow .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .371
38.4 Stopped Flow .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .372
38.5 Flow Kinetics .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .372
38.6 Temperature and Pressure Jumps .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .372
38.7 Caged Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .374
38.8 Surface Plasmon Resonance . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .374
38.8.1 Theory of SPR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .377
38.8.2 Practical Aspects. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .378
38.8.3 Surface Plasmon Coupled Fluorescence .. . .. . . . . . . . . . . . . . . . .379
38.8.4 Dual Polarisation Interferometry.. . . . . . . . . . .. . . . . . . . . . . . . . . . .380
38.9 Quartz Crystal Microbalance . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .380
39 Isotope Effects.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .383
40 Isotope Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .387
40.1 ADP/ATP Exchange.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .387
40.2 18 O-Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .387
40.3 Positional Isotope Exchange .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .388
Part VII Protein–Ligand Interactions
40.3.1 Structural Aspects of Protein–Protein Interactions . . . . . . . . .389
41 General Conditions for Interpretable Results . . . . . . . . . . . .. . . . . . . . . . . . . . . . .391


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xv

42 Binding Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .393
42.1 The L ANGMUIR-Isotherm: A Single Substrate Binding
to a Single Binding Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .393
42.2 Binding in the Presence of Inhibitors.. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .394
42.2.1 Competitive Inhibition . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .394
42.2.2 Non-competitive Inhibition . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .395
42.3 Affinity Labelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .396
42.3.1 Differential Labelling . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .397
43 Methods to Measure Binding Equilibria . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .399
43.1 Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .399
43.1.1 Equilibrium Dialysis . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .399
43.1.2 Continuous Dialysis . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .400
43.2 Ultrafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .400
43.3 Gel Chromatography .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .402
43.3.1 The Method of H UMMEL AND D REYER . . .. . . . . . . . . . . . . . . . .402
43.3.2 Spin Columns .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .402
43.4 Ultracentrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .403
43.4.1 The Method of D RAPER AND V. H IPPEL . .. . . . . . . . . . . . . . . . .403
43.4.2 The Method of S TEINBACH AND S CHACHMAN . . . . . . . . . . .403
43.5 Patch-Clamping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .404
43.6 Mass Spectrometry .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .405
43.7 Determination of the Number of Binding Sites:
The Job-Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .406

44 Temperature Effects on Binding Equilibrium
and Reaction Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .409
44.1 Activation Energy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .409
44.2 Isothermal Titration Calorimetry . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .412
44.2.1 Photoacoustic Calorimetry . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .413
Part VIII Industrial Enzymology
45 Industrial Enzyme Use .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .417
45.1 Enzyme Denaturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .419
45.2 Calculation of the Required Amount of Enzyme . . . .. . . . . . . . . . . . . . . . .420
46 Immobilised Enzymes .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .421
46.1 Kinetic Properties of Immobilised Enzymes .. . . . . . . .. . . . . . . . . . . . . . . . .422
46.1.1 Factors Affecting the Activity
of an Immobilised Enzyme.. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .422
46.1.2 The Effectiveness Factor . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .422
46.1.3 Maximal Effective Enzyme Loading . . . . . . .. . . . . . . . . . . . . . . . .423
0
Over Time. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .423
46.1.4 Decline of Vmax

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xvi

Contents

Part IX

Special Statistics


47 Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .427
47.1 Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .428
47.2 Assessing the Quality of Measurements . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .431
47.3 Analytical Results Need Careful Interpretation .. . . . .. . . . . . . . . . . . . . . . .432
47.4 False Positives in Large-Scale Screening . . . . . . . . . . . .. . . . . . . . . . . . . . . . .433
48 Testing Whether or Not a Model Fits the Data .. . . . . . . . . .. . . . . . . . . . . . . . . . .435
48.1 The Runs-Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .436
Part X

Appendix

A

List of Symbols .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .441

B

Greek Alphabets .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .445

C

Properties of Electrophoretic Buffers . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .447

D

Bond Properties .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .453

E

Acronyms .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .455


References .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .465
Index . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .487

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Part I

Analytical Techniques

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Chapter 1

Microscopy

The microscope is without question the most important instrument available to the
biologist. The physiological function of proteins cannot be addressed without taking their localisation in a living cell and their interaction with other proteins into
account. To answer these questions the microscope is an invaluable tool. http://
micro.magnet.fsu.edu/primer/ covers microscopic techniques in much more detail
than possible here.

1.1 Optical Foundations of Microscopy
Objects in cell biology range from an ostrich egg (20–30 cm) down to subcellular
organelles. An impression of this range is given by Fig. 1.1. Although some objects

can be seen with the naked eye, magnification is required for others.

ă
1.1.1 K OHLER
-Illumination
The microscope is a system of lenses which create images of the illumination and
object planes (see Fig. 1.2). In the illumination planes an image of the light source
is created; in the object planes an image of the object. As you can see from the
figure, light from each point of the light source is spread evenly across the entire
object planes, passing the object in parallel beams from every azimuth. This creates a homogeneous, bright illumination; thus only a low power light source (low
voltage halogen lamp of about 20 W) is required. The apertures have to be adjusted
in position and diameter to avoid contrast reduction by stray light and to achieve
maximum resolution.
1.1.1.1 Critical Illumination
Simpler microscopes with critical (N ELSON) illumination are used in lab classes
or clinical laboratories. In this case the image of the light source (the frosted glass
E. Buxbaum, Biophysical Chemistry of Proteins: An Introduction
to Laboratory Methods, DOI 10.1007/978-1-4419-7251-4 1,
© Springer Science+Business Media, LLC 2011

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3


4

1 Microscopy

Size of objects in cell biology


100 pm

H atom

X-ray
1 nm
globular protein
10 nm

visible light
IR light

1 um

Microwaves

10 um

100 um

Bacterium

animal cell
plant cell

light microscope

UV light


electron microscope

Virus, ribosome
100 nm

Radar

naked eye

1 mm

1 cm
Caulerpa

ultrashort waves (FM, TV)

10 cm
ostrich egg

Fig. 1.1 Size of objects in cell biology, compared to the wavelengths of different electromagnetic
waves

of a light bulb) is projected into the object plane by the condenser. Such microscopes are easier to use since condenser height and field diaphragm need not be
readjusted each time the objective is changed; they are also considerably cheaper.
With modern, multilayer-coated lens systems the increase in stray light and reduction in resolution is not relevant for Na Ä 0:5. For micro-photography, however, this
system is not suitable, since “hard” (high contrast) films amplify any inhomogeneity
in the illumination.

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1.1 Optical Foundations of Microscopy
Fig. 1.2 Schematic diagram
of a microscope with the
illumination system originally
introduced by AUGUST
ă
K OHLER
at Zeiss. The lens
systems create images of both
the object and the light
source. The illumination
apertures adapt the numerical
aperture of the illumination
system to that of the
objective. The apertures in
the object plane adjust the
field of view. Thus the
diameter of the light beam
and its opening angle can be
adjusted independently. This
reduces the stray light inside
the microscope. Careful
adjustment of the size and
position of the apertures is
required to take full benefit of
the microscope

5


Retina

Lens
Pupilla

}

Eye

Ocular
intermediate image
(in eye piece diaphragm)

Objective aperture
(virtual)
Objective

Object

Condenser

Condenser aperture

Field diaphragm

Collector

Light source
Mirror


1.1.2 The Role of Diffraction
Two factors influence the power of a microscope: resolution and contrast. Contrast
can nearly always be increased by staining or by optical methods (see later), the
microscope only needs to keep the level of stray light down. The resolution (the
minimal distance between two objects that still allows them to be seen as separate),
however, is subject to tight physical limitations.
Responsible for the image formation is the process of scattering the light waves
on the object. The scattered light creates a primary interference pattern for each
point of the condenser aperture in the objective aperture of the microscope (see
Fig. 1.3 on p. 6 for a brief discussion of diffraction). The distance between the
diffraction maxima depends on the grid constant (distance between structures) of
the object.

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6

1 Microscopy

a
parallel light beam

Edge

linear wave front

wall with pin hole

direction of light


b

curved wave front

c

plane wave front
wall with 2 pin holes

d

Intensity

curved wave fronts with interference pattern

−2

−1

0

+1

+2

position

Fig. 1.3 (a) If a light beam is passed through a pin hole, light beams at the edge of the hole
depart from their path. Simple beam optics can not explain such behaviour. (b) The same situation

viewed by wave optics. A linear wave front (equivalent to parallel light beams) reaches a wall with
a pin hole. Scattering results in a curved wave front. (c) If there are two pin holes, the resulting
wave fronts interfere with each other. Out-of-phase waves cancel, in-phase waves amplify each
other. Thus a pattern of bright and dark rings becomes visible. The central bright disk is called
the maximum of zeroth order, the surrounding rings are numbered first, second. . . order. (d) The
light intensity plotted as function of the position in the interference pattern. The maximum of
zeroth order is much brighter than the first, higher order maxima are even weaker. Note that the
interference rings are symmetrical, thus each ring results in two peaks, one to the right (positive
numbers) and one to the left (negative numbers) of the centre

Since the light from these diffraction maxima continues to travel upwards, and
since it comes from a single point, the beams are capable of interference. This
creates a secondary interference pattern, the intermediate image, which is then magnified by the ocular. An animated discussion of interference may be found at http://
www.doitpoms.ac.uk/tlplib/diffraction/index.php.

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1.1 Optical Foundations of Microscopy

7

The simplest object is a tiny hole. Its image is a pattern of bright and dark rings,
called A IRY-pattern.

1.1.3 The Importance of the Numerical Aperture Na
As originally found by E RNST A BBE [1] the intermediate image is the more similar
to the object, the more diffraction maxima are collected for it. The diffraction maximum of zeroth order contains most of the light, but no information, since it has not
interacted with the object. This means that to reconstruct the object in the intermediate image, at least the diffraction maximum of first order must be in the objective
aperture. Since finer object details give a higher distance of the refraction maxima

from each other, the resolution capability of a microscope depends on the objective
aperture.
The radius of this aperture is calculated to
r D sin.˛/=nm

(1.1)

with ˛ being the opening angle of the light cone and nm the refractive index of
the medium. Optically important media and their refractive indexes can be found in
Table 1.1.
On each objective you should find three numbers engraved: The magnification,
the mechanical tubus length for which the objective was calculated (usually either
160 mm or infinity) and the numerical aperture Na , with
Ã

Â

1
Na D sin
2

˛

nm

(1.2)

The point-spread function of an ideal (abberation-free) lens is an A IRY-pattern
(see Fig. 1.4):
!2

2J1 . 2 rNa /
h.r/ D
(1.3)
2 rN
a

Table 1.1 Refractive index n
of important media

Medium
Air
Water
Glycerol
Fused silica
Toluene
Glass
Immersion oil

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n
1.000
1.330
1.460
1.462
1.489
1.520
1.520



8

1 Microscopy
PSF (Airy pattern)
400 nm
700 nm

1

intensity

0.8
0.6
0.4
0.2
0
-1000 -800 -600 -400 -200

0

200 400 600 800 1000

radius

Fig. 1.4 Point-spread function of an ideal (abberation-free) lens. The diameter of the zeroth order
maximum (A IRY -disk) increases with , hence the possible resolution decreases

with r the radius and J1 . / the B ESSEL-function of first kind and first order:
1
X


. 1/m
Ja . / D
mŠ .m C a C 1/
mD0

 Ã2mCa
2

(1.4)

The -function is a generalisation of the faculty-function for real arguments.
Since J1 . / has its first minimum at = 3.83, the radius of the first minimum of
the A IRY-pattern is
0:61
(1.5)
rD
Na
The Na of both the condenser and the objective determine the obtainable resolution
d . The radius of the A IRY-disk of a point is
rAiry D

0:61
Na .objective/ C Na .condenser/

(1.6)

We can imagine that each point of an object results in its own A IRY-pattern, resulting in a mosaic image. Experience shows that two points are seen as separate if
the distance between their A IRY-disks is at least equal to rAiry , then the minimum
between the overlapping disks has about 80 % of the intensity of the maxima. However, due to optical imperfections the radius of the A IRY-disk is somewhat larger

than that calculated above, empirically a factor of 112 % is assumed.
Na also determines the light flux ˚, the axial resolution ra (which is considerably
worse than the lateral resolution), and the depth of focus T :
˚ D ˚0

 

Na

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(1.7)